1a.Objectives (from AD-416)
To discover phytotoxins and allelochemicals for use in pest management and to provide the fundamental information on mode of action, structure-activity relationships, natural resistance mechanisms, and biosynthesis of natural phytotoxins that will be required for development medicinal/nutraceutical crops. To discover, characterize, manipulate and utilize genes involved in the production of natural products and in chemical defense and resistance mechanisms against allelochemicals and environmental phytotoxins.

1b.Approach (from AD-416)
Conduct bioassays in collaboration with research chemists during bioassay-directed isolation of new phytotoxins. Molecular sites of action will be determined with genomic and biochemical approaches. Biosynthetic pathways of toxins from plants will be investigated. Genes controlling synthesis of useful plant secondary products and plant defenses to phytotoxins will be identified, cloned, and manipulated.

3.Progress Report
The main thrusts of this project have been to discover new, natural product-based weed management tools. We have discovered several highly active natural products that could be used as herbicides or bases for synthetic herbicides. Generation of a transcriptome data base for identification of new herbicides target sites is almost completed. We are in the final phases of transgenic manipulation of sorgoleone, a potent allelochemical in sorghum species.

Dose-response curves of a set of natural ß-triketones (e.g., flavesone, grandiflorone and leptospermone) and several analogues against plant HPPD were obtained. Several of the triketone tested were extremely potent against HPPD. The steric and electrostatic contributions were determined using conformational molecular field analysis (CoMFA) and the hydrophobic contribution was analyzed with hydropathic interaction (HINT). Bulky substituents on the triketone ring structure reduced the overall inhibitory activity of the compounds whereas increase in the aliphatic tail improved the activity of the compounds up to a point. Modeling of the binding of the triketones to HPPD and computational analysis of the catalytic domain of HPPD provided a structural basis to explain the structure-activity relationship revealed by CoMFA and HINT.

Sorgoleone is a potent natural herbicide (allelochemical) produced by sorghum (Sorghum bicolor) root hairs. Sorgoleone is a strong inhibitor of photosynthesis and it may also act on other target sites, such as p-hydroxyphenylpyruvate dioxygenase and membrane localized proton pumps. However, most of these experiments were done in the laboratory and under in vitro conditions. This research investigates how sorgoleone act on whole plants. We discovered that sorgoleone can act as a photosynthetic inhibitor on very young plants but it has little effect by this mechanism on plants 7-days or older. Furthermore, sorgoleone disrupts plant cell membrane integrity in the dark, which involves processes independent of photosynthesis. Therefore, sorgoleone has at least two mechanisms of herbicidal action.

3.
Characterization of the role of antioxidants in the protection of plants against natural and synthetic inhibitors of protoporphyrinogen oxidase.

Concentrations of the photodynamic molecule protoporphyrin IX (Proto IX ) increase dramatically in the presence of protoporphyrin oxidase (protox) inhibitors (e.g., acifluorfen). In the presence of light, this causes the formation of highly reactive oxygen species responsible for the loss of cellular membrane integrity and death of plants. Some plants species are much less susceptible to these herbicides than others and it has been suggested that plants with high antioxidant contents are less sensitive to inhibition of protox. We show that increasing the levels of certain antioxidants does protect plants against the herbicidal effect of acifluorfen. In particular, hydrophilic antioxidants such as glutathione and ascorbic acid had superior protective effect than the lipophilic antioxidant tocopherol. Conversely, inhibiting glutathione biosynthesis with 5 mM L-buthionine-sulfoximine rendered plants more sensitive to AFM.

4.
Development of transgenic sorghum deficient in sorgoleone biosynthesis using RNAi technology

In previous reports we described the functional characterization of two polyketide synthase (PKS) sequences isolated from a root hair EST data set developed by our research unit. These sequences were found to encode alkylresorcinol synthase-type PKSs based on their ability to accept long- and medium-chain acyl-CoA starter substrates and catalyze the formation alkylresorcinols – indicating a potential role for these enzymes in sorgoleone biosynthesis. A strategy was therefore devised using RNA interference (RNAi) technology to test whether downregulation of these PKS sequences would result in the generation of sorghum plants deficient in the production of the allelochemical sorgoleone. Eight independent transformed sorghum lines harboring transgenes designed for the simultaneous RNAi-mediated inhibition of both PKS genes were analyzed. All of the RNAi lines were found analyzed for successful transformation and sorgoleone production. An absolute correlation was found between successful transformation and loss of detectable amounts of sorgoleone and PKS transcript levels. Thus, a major milestone was achieved for this project in our ability to manipulate sorgoleone levels in planta using genes isolated by our research unit from a database developed in-house.

Databases of transcriptome signatures for compounds with known modes of action can serve as important resources for obtaining clues concerning the mode of action of unknown bioactive compounds. Toward this end, we have used Arabidopsis as a model for generating whole-genome transcriptome responses. Our system more closely emulates conditions for plants exposed to herbicides under field conditions than studies performed by other laboratories. Using this system, detailed transcriptome responses for plants exposed to herbicides targetting auxin, acetolactate synthase, EPSP synthase, photosystem II, and photosystem I have been generated for plants exposed to both sublethal and lethal levels, analyzed at 4, 24, and 48 hours post-treatment. Our data set now spans the majority of herbicide modes of action associated with chemistries amenable to post-emergence herbicide application.